A Versatile Processing Workflow to Enable Pathogen Detection in Clinical Samples from Organs Using VIDISCA

Diagnostics

Alba Folgueiras-González¹  Robin van den Braak¹  Ad de Groof¹  Lia van der Hoek²  Martin Deijs² 

[1] Department Discovery & Technology, MSD Animal Health, Wim de Körverstraat 35, P.O. Box 31, 5830AA Boxmeer, The Netherlands;Laboratory of Experimental Virology, Department of Medical Microbiology, Amsterdam UMC location AMC, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands;

关键词: virus discovery;    metagenomics;    VIDISCA-NGS;    atypical porcine pestivirus;    enrichment;   

DOI  :  10.3390/diagnostics11050791

来源: DOAJ

【 摘 要 】

In recent years, refined molecular methods coupled with powerful high throughput sequencing technologies have increased the potential of virus discovery in clinical samples. However, host genetic material remains a complicating factor that interferes with discovery of novel viruses in solid tissue samples as the relative abundance of the virus material is low. Physical enrichment processing methods, although usually complicated, labor-intensive, and costly, have proven to be successful for improving sensitivity of virus detection in complex samples. In order to further increase detectability, we studied the application of fast and simple high-throughput virus enrichment methods on tissue homogenates. Probe sonication in high EDTA concentrations, organic extraction with Vertrel™ XF, or a combination of both, were applied prior to chromatography-like enrichment using Capto™ Core 700 resin, after which effects on virus detection sensitivity by the VIDISCA method were determined. Sonication in the presence of high concentrations of EDTA showed the best performance with an increased proportion of viral reads, up to 9.4 times, yet minimal effect on the host background signal. When this sonication procedure in high EDTA concentrations was followed by organic extraction with Vertrel™ XF and two rounds of core bead chromatography enrichment, an increase up to 10.5 times in the proportion of viral reads in the processed samples was achieved, with reduction of host background sequencing. We present a simple and semi-high-throughput method that can be used to enrich homogenized tissue samples for viral reads.

【 授权许可】

Unknown